Methods and compositions for treating Raynaud&#39;s Phenomenon and scleroderma

ABSTRACT

A method for treating conditions or diseases associated with deleterious vasoconstriction of the small arteries and arterioles of one or more organs or parts of a patient&#39;s body. In one embodiment, the method comprises administering a therapeutically effective amount of an antagonist to the α 2C -adrenergic receptor (α 2C -AR) to a patient with Raynaud&#39;s Phenomenon. Such method is used to ameliorate the cold-induced or stress-induced vasoreactive response that is associated with Raynaud&#39;s Phenomenon. The α 2C -AR antagonist is administered to the subject either prior to or after exposure of the patient to the cold or to stressIn another embodiment, the method is used to reduce the extent of deleterious vasoconstriction that occurs in the small arteries, arterioles, and microcirculation of the lungs, heart, kidneys, skin, or gastrointestinal tract of a patient, particularly a scleroderma patient. The method comprises administering a therapeutically effective amount of an α 2C -AR antagonist to a patient who is in need of the same. Such treatment serves to maintain or restore, at least in part, blood flow through the small arteries, arterioles, and microcirculation of the lungs, heart, kidneys, skin, and/or gastrointestinal tract in a such patient. The present invention also relates to a pharmaceutical compositions comprising an α 2C -AR antagonist and a pharmaceutically acceptable carrier.

This work was supported, at least in part, by a grant from the NationalInstitutes of Arthritis and Musculo-Skeletal and Skin Diseases Grant No:AR-46126. The United States Government has certain rights in thisinvention.

BACKGROUND

Raynaud's Phenomenon is one example of a disease that involvesdeleterious vasoconstriction of the small arteries and/or arterioles ofone or more organs of a subject's body. Raynaud's Phenomenon is anabnormal vasoreactive response to cold or emotional stress of the smallarteries and arterioles in the subject's digits. Individuals who sufferfrom Raynaud's Phenomenon experience episodic, sharp, demarcated,cutaneous pallor and cyanosis of their digits. These symptoms resultfrom spasm or closure of the digital arteries. The condition is painfuland debilitating. Under severe conditions, it can even lead to digitalulcers or amputation of the affected digit.

Another, more problematic disease that is associated with a deleteriousvasoconstriction of the small arteries and arterioles in one or moreorgans of a subject's body is Scleroderma. Scleroderma is a devastatingdisease of unknown etiology or origin that is associated with severemorbidity and mortality. Vascular dysfunction is an important earlydefect in Scleroderma (SSc). Raynaud's phenomenon is one of the earliestmanifestations of SSc, occurring in approximately 95% of patients. Inaddition to the digital arteries, reversible vasospasm also occurs inthe terminal arterial supply of the kidney, heart, lung, andgastrointestinal tract of patients with scleroderma. Such vasospasticactivity causes ischemia, reperfusion injury and increased oxidantstress of the affected organs and is thought to thereby contribute toendothelial injury and the vascular and extravascular lesions thatsubsequently occur in this disease. The vascular lesions are found inthe small arteries, arterioles (50-500μ in diameter) and themicrocirculation of the affected organ and are characterized byconcentric intimal thickening and adventitial fibrosis of the smallarteries and arterioles. The loss of function and structure of theaffected blood vessels leads to ischemia of the organ supplied by thesevessels, organ failure, and death.

At present there is no cure and no effective therapy for the diseasesthat involve deleterious vasoconstriction of the small arteries andarterioles, including scleroderma. In addition, there is no therapy thatis specifically targeted to Raynaud's phenomenon. Current therapy forthis condition is limited to broad spectrum vasodilator therapy whichaffects every blood vessel of the treated individual and, thus, causessignificant side effects, such as dizziness, nausea, and severeheadaches, vasodilator therapy could also exacerbate the problem bydirecting blood away from the affected organ.

Accordingly, it is desirable to have new methods and pharmaceuticalcompositions which can be used to treat diseases that involvedeleterious vasonstriction of the small arteries and arterioles,including Raynaud's Phenomenon and scleroderma. Methods andpharmaceutical composition which do not cause systemic vasodilation areespecially desirable.

SUMMARY OF THE INVENTION

The present invention provides methods for treating diseases associatedwith deleterious vasoconstriction of the small arteries and arteriolesof one or more organs or parts of a patient's body. In one embodiment,the method comprises administering a therapeutically effective amount ofan antagonist to the α_(2C)-adrenergic receptor (α_(2C)-AR) to a patientwith Raynaud's Phenomenon. Such method is used to ameliorate thecold-induced or stress-induced vasoreactive response that is associatedwith Raynaud's Phenomenon. The α_(2C)-AR antagonist is administered tothe subject either prior to or after exposure of the patient to the coldor to stress. Preferably, the antagonist is administered orally or in atopical composition. To prevent or reduce the extent of thevasoconstriction that occurs when such patient is exposed to stress orcold, the α_(2C)-AR antagonist is administered to the patient prior tosuch exposure. Such treatment serves to maintain, at least in part,blood flow through the cutaneous microcirculation of a patient withprimary or secondary Raynaud's phenomenon. To reverse or lessen the coldinduced or stress-induced vasoconstriction of the cutaneous arterialcirculation in such patient, the α_(2C)-AR antagonist is administered tothe patient after exposure to the cold or to the stress. Such treatmentserves to restore, at least partially, blood flow through the cutaneousarterial circulation of the treated patient.

In another embodiment, the method is used to reduce the extent ofdeleterious vasoconstriction that occurs in the small arteries,arterioles, and microcirculation of the lungs, heart, kidneys, skin, orgastrointestinal tract of a patient, particularly a scleroderma patient.The method comprises administering a therapeutically effective amount ofan α_(2C)-AR antagonist to a patient who is in need of the same. Suchtreatment serves to maintain or restore, at least in part, blood flowthrough the small arteries, arterioles, and microcirculation of thelungs, heart, kidneys, skin, and/or gastrointestinal tract in a suchpatient.

The present invention also provides methods and compositions forstudying vasoconstriction in other disease states such as pulmonaryhypertension, renal ischemia, gastrointestinal ischemia, and coronaryischemia hypertension, and for studying the physiology ofvasoconstriction.

The present invention also relates to a pharmaceutical compositionscomprising an α_(2C)-AR antagonist and a pharmaceutically acceptablecarrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure of the α_(2C)-AR antagonist, MK 912.

FIG. 2 shows the vasoconstrictor effects of the selective α₁-AR agonist,phenylephrine (10⁻⁹ to 3×10⁻⁷M), or the selective α₂-AR agonist, UK14,304 (10⁻⁹ to 3×10⁻⁷M), in proximal and distal tail arteries of themouse. Vasoconstriction was expressed as a percentage of the stablebaseline diameter and is presented as means±SEEM (n=4 [UK 14,304] or 3[phenylephrine]).

FIG. 3 shows the effect of cold (from 37° C. to 28° C.) on thevasoconstrictor response to the selective α₁-AR agonist, phenylephrine(10⁻⁹ to 10⁻⁶M), in distal tail arteries of the mouse. Vasoconstrictionwas expressed as a percentage of the stable baseline diameter and ispresented as means±SEM (n=4).

FIG. 4 shows the effect of cold (from 37° C. to 28° C.) on thevasoconstrictor response to the selective α₂-AR agonist, UK 14,304 (10⁻⁹to 3×10⁻⁷M), in distal tail arteries of the mouse. Vasoconstriction wasexpressed as a percentage of the stable baseline diameter and ispresented as means±SEM (n=4).

FIG. 5 shows the effect of the α_(2A)-AR antagonist, BRL 44408 (100 and1,000 nM), on the vasoconstrictor response to the α₂-AR agonist, UK14,304 (10⁻⁹ to 3×10⁻⁷M), in distal tail arteries of the mouse.Inhibitory effect of BRL 44408 was assessed at 37° C. (warm, upperpanel) and at 28° C. (cold, lower panel). Vasoconstriction was expressedas a percentage of the stable baseline diameter and is presented asmeans±SEM (n=4). Absence of error bar indicates the SEM was less thanthe size of the symbol.

FIG. 6 shows the effect of the α_(2C)-AR antagonist, MK 912 (0.3 nM), onthe vasoconstrictor response to the α₂-AR agonist, UK 14,304 (10−9 to3×10⁻⁷M), in distal tail arteries of the mouse. Inhibitory effect of MK912 was assessed at 37° C. (warm, upper panel) and at 28° C. (cold,lower panel). Vasoconstriction was expressed as a percentage of thestable baseline diameter and is presented as means±SEM (n=4).

FIG. 7 shows the effect of the α_(2C)-AR antagonist, MK 912 (0.3 nM), oncold-induced augmentation of α₂-AR vasoconstriction in distal tailarteries of the mouse. Vasoconstrictor responses to the α₂-AR agonist,UK 14,304 (10⁻⁹ to 3×10⁻⁷M) were assessed as described in FIG. 4.However, in contrast to FIG. 4, α_(2C)-AR s were blocked by treating thearteries with MK 912 (0.3 nM) before and during each of theconcentration effect curves. Vasoconstriction was expressed as apercentage of the stable baseline diameter and is presented as means±SEM(n=4).

FIG. 8 shows the vasoconstrictor responses evoked by the selective α₂-ARagonist UK 14,304 (panel A) the selective α₁-AR agonist phenylephrine(panel B), or the receptor-independent stimulus KCl (panel C) in controland SSc arterioles. Concentration-effect curves were obtained inarterioles with endothelium (circles). In addition, the response to UK14,304 (100 nM, log M of −7) is also presented for endothelium-denudedarterioles (squares). Empty symbols, control; filled symbols, SSc.Concentration is expressed as the molar concentration (moles/liter, M)of the agonist in the solution and presented as log M. SSc arterioleshad increased reactivity to α₂-AR stimulation demonstrated by anincreased maximal response to the agonist (P=0.000014) and increased AUC(P<0.00037). In contrast, constrictor responses to KCl or to the α₁-ARagonist, phenylephrine were not significantly different between controland SSc arterioles (KCl maximal observed response 60 mM: P=0.44, AUC:P=0.36; Phenylephrine maximal observed response 1 μM: P=0.87, AUC:P=0.23)

FIG. 9 shows the levels of mRNA molecules encoding the α_(2A) and theα_(2C) adrenergic receptors in the dermal arteries of healthy controlsubjects and patients with scleroderma.

FIG. 10 shows the effect of the α_(2C)-AR antagonist, MK 912 (0.3 nM),on the vasoconstrictor response to the α₂-AR agonist, UK 14,304 (10⁻⁹ to3×10⁻⁷M), in dermal arteries of healthy control subjects and patientswith scleroderma.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating diseases whichinvolve a deleterious vasoconstriction of the small arteries and/orarterioles of at least one organ or part of a patient's body. Suchdiseases include Raynaud's Phenomenon and scleroderma. Such methodscomprise administering a therapeutically effective amount of anα_(2C)-AR antagonist to a patient in need of the same. As used hereinthe term “α_(2C)-AR antagonist” refers to a compound that selectivelybinds to an α_(2C) adrenergic receptor, i.e., the binding affinity ofthe compound to the α_(2C) adrenergic receptor is at least three timesgreater than the binding affinity of the compound to the α_(2A)adrenergic receptor or the α_(2B) adrenergic receptor. In other words,the K_(D) of the antagonist and α_(2C)-ARs is at least 3-fold lower thanthe K_(D) of the antagonist and α_(2A)-ARs or α_(2B)-ARs. Furthermore,the α_(2C)-AR antagonist, when given at the same concentration, has theability to block the α₂-AR-related response of vascular smooth musclecells treated with an α₂-AR agonist. Preferably, binding of theantagonist to the α_(2C)-AR is reversible. Preferably, the antagonist isa competitive inhibitor of the α₂-AR agonist. One example of a suitableα_(2C)-AR antagonist is 1′, 3′-dimethylspiro (1, 3, 4, 5′, 6, 6′, 7,12b-octahydro-2H-benzo[b]furo[2,3-a) quinolizine-2,4′-pyrimidin-2′-one,which is available from Merck Chemical Company and has the structureshown in FIG. 1.

Unlike α₁-ARs, functional α₂-ARs are not widely distributed in thevascular system. α₂-AR constrictor activity is not present in largearteries, but is generally restricted to small arteries/arterioles andto the venous circulation. α₂-ARs are known to comprise three subtypes;α_(2A), α_(2B), and α_(2C). Although the α₂-AR subtypes are highlyhomologous (50-60% amino acid identity), they are uniquely sensitive tophysiological regulation. α_(2C)-ARs have a predominantly intracellulardistribution (endoplasmic reticulum and golgi apparatus) whereasα_(2A)-AR are localized on the cell membrane. Furthermore, thesereceptor subtypes are reported to have differing sensitivities todesensitization and in their efficiency of coupling to G-proteins.

FORMULATION

The pharmaceutical composition comprises an α_(2C)-AR antagonist and apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the activity of the α_(2C)-AR antagonist. Thecharacteristics of the carrier will depend on the route ofadministration. The pharmaceutical composition, which comprises theα_(2C)-AR antagonist and carrier, optionally further contains otheragents which either enhance the activity of α_(2C)-AR antagonist orcomplement its activity in treating the condition or disease.Optionally, additional factors and/or agents are included in thepharmaceutical composition to minimize side effects of the α_(2C)-ARantagonist. Optionally, the pharmaceutical composition containsdiluents, fillers, salts, buffers, stabilizers, solubilizers,antioxidants, preservatives and other materials which are conventionallyused in pharmaceutical compositions.

Optionally, the pharmaceutical composition is in the form of a liposomein which α_(2C)-AR antagonist is combined with amphipathic agents suchas lipids which exist in aggregated form as micelles, insolublemonolayers, liquid crystals, or lamellar layers in aqueous solution.Suitable lipids for liposomal formulation include, without limitation,monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,saponin, bile acids, and the like. Preparation of such liposomalformulations is conventional.

Routes of Administration

Administration of a pharmaceutical composition comprising the α_(2C)-ARantagonist is carried out in a variety of conventional ways, such asoral ingestion, inhalation, topical application, or cutaneous,subcutaneous, intramuscular, intraperitoneal, parenteral or intravenousinjection. For Raynaud's Phenomenom, local administration of a topicalcomposition to the affected site or oral ingestion is preferred. Forscleroderma or other diseases which involve deleterious vasoconstrictionof the microcirculation of the lungs, kidneys, or gastrointestinaltract, the preferred route of administration is oral ingestion.

When the α_(2C)-AR antagonist is administered orally, the pharmaceuticalcomposition is, preferably, in the form of a tablet, capsule, powder,solution or elixir. When administered in tablet form, the pharmaceuticalcomposition of the invention optionally contains a solid carrier such asa gelatin or an adjuvant. When administered in liquid form, a liquidcarrier such as water, petroleum, oils of animal or plant origin such aspeanut oil, mineral oil, soybean oil, or sesame oil, corn oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition optionally contains physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol.

When the α_(2C)-AR antagonist is administered by intravenous,intramuscular, intraperitoneal, parenteral, cutaneous or subcutaneousinjection, the pharmaceutical composition is in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof a parenterally acceptable aqueous solution, having suitable pH,isotonicity, and stability, is conventional. A preferred pharmaceuticalcomposition for intravenous, intramuscular, cutaneous, or subcutaneousinjection, preferably, contain, in addition to α_(2C)-AR antagonist, anisotonic vehicle such as Sodium Chloride Injection, Ringer's Injection,Dextrose Injection, Dextrose and Sodium Chloride Injection, LactatedRinger's Injection, or other vehicle.

Preferably, intravenous therapy is used when the patient, particularlythe scleroderma patient, is in crisis. Intravenous administration,preferably, is continued until the crises subsides and the patient canbe switched to oral administration. The duration of intravenousadministration depends on the severity of the crises, the condition ofthe individual patient, and the response of each individual patient tothe intravenous administration.

Dosage

The α_(2C)-AR antagonist is administered to the patient in atherapeutically effective amount. As used herein, the term“therapeutically effective amount” means the total amount of α_(2C)-ARantagonist that is sufficient to show a meaningful benefit, i.e.,treatment, healing, prevention, reversal or amelioration of thecondition or disease, or an increase in rate of treatment, healing,prevention or amelioration of such disease. The amount of α_(2C)-ARantagonist administered to the patient depends upon the nature andseverity of the condition being treated, the mode of administration, andon the nature of prior treatments which the patient has undergone. Thedosages of α_(2C)-AR antagonist used to treat the condition or diseaseare determined by running routine trials with appropriate controls. Insuch studies, varying dosages of the α_(2C)-AR antagonist areadministered indirectly to the subject or directly to the affectedtissue or organ and the amount of antagonist sufficient to reduce,reverse, or prevent vasoconstriction and/or to improve blood flowthrough the small arteries, arterioles, and microcirculation of theaffected digit or organ under conditions which cause restricted bloodflow, such as cold or stress, is determined. Preferably, the dosage usedis sufficient to return blood flow in the affected tissues or organs tolevels comparable to those levels found in the same type of tissue ororgan from control subjects, e.g., subjects that do not have Raynaud'sPhenomenon or scleroderma. Preferably, the pharmaceutical compositioncontains from about 0.01 μg to about 100 mg, more preferably about 0.01μg to about 10 mg, most preferably about 0.1 μg to about 1 mg of theα_(2C)-AR per kg body weight.

Although a single dose of the α_(2C)-AR antagonist may be sufficient toameliorate the pathological effect of the condition or disease and toreturn blood flow levels to normal or near normal, it is expected thatmultiple doses of the α_(2C)-AR antagonist will be administered to thepatient, particularly to patients with scleroderma.

Population Receiving Treatment

The α_(2C) AR antagonist is administered to a patient who has exhibitedsymptoms of or has been diagnosed with a disease or condition associatedwith a deleterious vasoconstriction of one or more parts or organs ofthe patient's body. In the case of Raynaud's Phenomenon, the α_(2C)-ARantagonist is administered to a patient diagnosed as having Raynaud'sPhenomenon before or after the patient is exposed to the cold oremotional stress. In the case of scleroderma, administration of theantagonist, preferably, is begun as soon as possible followingdiagnosis. Preferably the α_(2C)-AR antagonist is administered to suchpatients throughout their lifetime in order to prevent further lesionsor deleterious changes to the affected or involved organs.

EXAMPLES

The following examples are for purposes of illustration only and are notintended to limit the scope of the invention as defined in the claimswhich are appended hereto.

Example 1

Inhibition of Cold-Augmented Vasoconstriction of Cutaneous Arteries byan α_(2C)-AR Antagonist

To determine the role of α₂-AR subtypes in cold-induced vasoconstrictionand the effect of selective α₂-AR antagonists on this phenomenon, a newmodel of the cutaneous circulation, namely the mouse tail artery, wasused.

Methods and Materials

Blood Vessel Chamber

Male mice (C57BL6) were euthanized by CO₂ asphyxiation. Proximal and/ordistal segments of tail artery were then rapidly removed and placed incold Krebs-Ringer bicarbonate solution (in mM): 118.3 NaCl, 4.7 KCl, 1.2MgSO₄, 1.2 KH₂PO₄, 2.5 CaCl₂, 25.0 NaHCO₃, 11. glucose (controlsolution). The small arteries were cannulated at both ends with glassmicropipettes, secured using 12-0 nylon monofilament suture and placedin a microvascuiar chamber (Living Systems, Burlington, Vt.). Thearteries were maintained at a constant transmural pressure of 60 mmHg inthe absence of flow. The chamber was superfused with control solutionand maintained at 37° C., pH 7.4, and gassed with 16% O₂-5% CO₂-balanceN₂. The chamber was placed on the stage of an inverted microscope (X20,Nikon TMS-F, Japan) connected to a video camera (Panasonic, CCTV camera,Japan). The vessel image was projected onto a video monitor and theinternal diameter continuously determined by a video dimension analyzer(Living Systems Instrumentation, Burlington Vt.) and monitored using aBIOPAC (Santa Barbara, Calif.) data acquisition system (GatewayDimensions Pentium Computer).

Protocol

Small arteries were allowed to equilibrate for 30-40 min at a transmuralpressure (PTM) of 60 mmHg before addition of α₁ and α₂-AR agonists andantagonists. Concentration-effect curves to the selective α₁-AR agonist,phenylephrine, or the selective α₂-AR agonist, UK 14,304 (brimonidine,5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) weregenerated by increasing the concentration of the agonists in half-logincrements, once the constriction to the previous concentration hadstabilized. Following completion of the concentration-effect curve, theinfluence of the agonists was terminated by repeatedly exchanging thebuffer solution and allowing the artery to return to its stable baselinelevel.

To evaluate the effect of selective α₂-AR antagonists onvasoconstriction, concentration-effect curves to UK 14,304 weredetermined under control conditions and in the presence of the selectiveα_(2A)-AR antagonist, BRL 44408 (100 and 1000 nM), the selectiveα_(2B)-AR antagonist ARC 0239 (50 nM) or the selective α_(2C)-ARantagonist MK 912 (0.3 nM) (Table 1). When these receptor antagonistswere used, the preparations were incubated for 30 min with the drugsprior to, and also during, exposure of the arteries to the agonists.When analyzing the influence of cold on α-AR responsiveness, thetemperature of the superfusate was decreased to 28° C. for 30 min priorto commencing a concentration effect curve to the constrictor agonists.This provides sufficient time for the effect of cold on adrenergicreactivity to stabilize.

Drugs

ARC 0239 was a gift from Boehringer Ingelheim (Ridgefield, Conn.), BRL44408 was a gift from SmithKline Beecham (Harlow, UK), MK 912 was a giftfrom Merck (West Point, Pa.), phenylephrine and sodium nitroprussidewere obtained from SIGMA (St. Louis, Mo.) and UK 14,304 was fromResearch Biochemicals International (Natick, Mass.). Stock solutions ofdrugs were prepared fresh each day and stored at 4° C. during theexperiment. Drugs were dissolved in distilled water with the exceptionof i) UK 14,304 which was dissolved in DMSO (highest chamberconcentration of 0.001%), ii) BRL 44408 which was dissolved in 0.1 N HCI(highest chamber concentration of 0.04%), iii) ARC 0239 which wasdissolved in methanol (highest chamber concentration of 0.004%). Atthese concentrations, the solvents did not alter reactivity of the bloodvessels. All drug concentrations are expressed as final molarconcentration (M, moles/liter) in the chamber superfusate.

Data Analysis

Vasomotor responses were expressed as a percentage change in internaldiameter (ID) prior to administrating the agent. Because of the phasicbehavior of the vasomotion in distal. tail arteries, the signal waselectronically averaged (BIOPAC software, smoothing factor of 2000) inorder to obtain diameter measurements. Functional data is expressed asmeans±SEM for n number of experiments, where n equals the number ofanimals from which blood vessels were studied. Antagonist dissociationconstants (K_(D)) were determined either from Arunlakshana and Schildplots or according to the formula: K_(D)=[Ant]/(CR-1), where [Ant] isthe concentration of antagonist, and CR the ratio ofagonist-concentrations producing equal responses in the presence andabsence of the antagonist. In all cases, slopes of Arunlakshana andSchild plots were not significantly different from unity, consistentwith competitive antagonism. Statistical evaluation of the data wasperformed by Student's t-test for either paired or unpairedobservations. When more than two means were compared, analysis ofvariance was used. If a significant F value was found, Scheffe's testfor multiple comparisons was employed to identify differences amonggroups. Values were considered to be statistically different when P wasless than 0.05.

Results

Baseline Characteristics

When transmural pressure (P_(TM)) was increased from 10 mmHg to 60 mmHg,distal segments of the mouse tail artery immediately dilated and thengradually constricted. The pressure-induced constriction or myogenicresponse comprised both tonic and phasic components. Administration ofthe vasodilator, sodium nitroprusside (10⁻⁵M ), abolished bothconstrictor components. Under these conditions, an increase intransmural pressure caused only a passive increase in arterial diameter(FIG. 2). In contrast to the distal segments, proximal segments of themouse tail artery did not constrict in response to increases in P_(TM)and, under quiescent conditions did not dilate to sodium nitroprusside(10⁻⁵M), indicating the absence of myogenic tone. These responses arecharacteristic of arterioles. Once the blood vessels had stabilized at60 mmHg, the internal diameter (ID) was 333.7±9.0μ (n=5) in proximal and157.8±14.8p (n=5) in distal segments of the mouse tail arteries.

α₁ and α₂-AR Activation

Stimulation of α₁-ARs by phenylephrine (10⁻⁹ to 3×10⁻⁷M) or α₂-ARS by UK14,304 (10⁻⁹ to 3×10⁻⁷M) caused concentration-dependent constriction ofthe proximal and distal segments of the mouse tail arteries (FIG. 2).Distal segment were significantly more responsive to α₂-AR activationbut significantly less responsive to activation of α₁-ARs, compared toproximal segments (FIG. 2). Thus, the constrictor activity of α₂-ARsincreased in distal compared to proximal segments, whereas the oppositepattern was observed for α₁-ARs. These are similar to results observedin human digital circulation. These results are consistent with previousreports that constrictor α_(2A)-ARs are functional in themicrocirculation, whereas in large arteries, the receptors are expressedbut not functional.

Influence of Cold on α-AR Constriction

The influence of cold on adrenergic constrictor responsiveness wasevaluated on distal segments. Cold did not significantly affect thebaseline diameter or myogenic tone in distal segments of the mouse tailarteries (IDs of 153±12.48 and 157.9±14.6p, at 37° C. and 28° C.,respectively, n=8). Furthermore, cold did not affect the constrictorresponse to stimulation of α₁-ARs by phenylephrine (FIG. 3). However,cold dramatically and reversibly increased the vasoconstriction causedby activation of α₂-ARs with UK 14,304 (FIG. 4).

Cold and α₂-AR Subtypes

At warm temperatures, vasoconstriction to the α₂-AR agonist UK 14,304was inhibited by the selective α_(2A)-AR antagonist BRL 44408 (100 nMand 1000 nM) (FIG. 5), but not inhibited by the selective α_(2B)-ARantagonist ARC 239 (50 nM) (data not shown) or the selective α_(2C)-ARantagonist MK 912 (0.3 nM) (FIG. 6). Based on the dissociation constants(K_(D)) for ARC 239 and MK 912 (Table 1), these antagonists would beexpected to cause ˜10-fold and ˜6-fold shifts in concentration-effectscurves generated by α_(2B)-AR and α_(2C)-AR stimulation, respectively.The Arunlakshana and Schild plot or the inhibitory effect of BRL 44408generated a −log K_(D) of 7.69±0.13 (K_(D of) 20 nM, n=4), consistentwith antagonism of α_(2A)-ARs (table 1). These results indicate that, atwarm temperatures, α_(2A)-ARs but not α_(2B)-ARs or α₂-ARs contribute toα₂-AR vasoconstriction.

During exposure to cold, the augmented vasoconstrictor response to UK14,304 was dramatically inhibited by the α_(2C)-AR antagonist MK912(3×10⁻¹⁰M) (FIG. 6). The inhibitory effect generated a −log K_(D) valueof 10.9±0.17 (K_(D) of 14 pM, n=4), consistent with inhibition ofα_(2C)ARs (Table 1). Inhibition of α_(2C)-ARs attenuated theα₂-AR-induced vasoconstriction only at low temperatures. Thus theα_(2C)-AR antagonist, MK 912, selectively abolished cold-inducedamplification of the α₂-AR response (FIG. 7).

The α₂-AR-induced vasoconstrictor response which occurred at 28° C. wasnot inhibited by the α_(2B)-AR antagonist ARC 0239 (50 nM, data notshown), but was reduced by the α_(2A) antagonist BRL 44408 (100 and 1000nM) (FIG. 5). The Arunlakshana and Schild plot for the inhibitory effectof BRL 44408 generated a −log K_(D) of 7.54+0.10 (K_(D) value of 29 nM,n=4), which was not significantly different from that observed at 37° C.Thus, blockade of α_(2A)-ARs with BRL 44408 inhibited α₂-AR-inducedconstriction to a similar degree at warm and cold temperatures, and didnot reduce the, cold-induced amplification of the response (compare theconcentration-effect curves in FIG. 6).

TABLE 1 K_(D) values (in nM) for α₂-AR antagonists Mouse Tail Arteryα-_(2A) ^(A) α-2B^(A) α-2C^(A) 37° C. 28° C. BRL44408 13 174 187 20 29(α-_(2A)) ARC239 256 4.6 51 >50 >50 (α-2B) MK912 1.8 .330.045 >0.3 >0.014 (α-2C)

^(A)Data from Flavahan, N. A., T. J. Rimele, J. P. Cooke, and P. M.Vanhoune. 1984. Characterization of postjunctional alpha-1 and alpha-2adrenoceptors activated by exogenous or nerve-released norepinephrine inthe canine saphenous vein. J Pharmacol Exp Ther. 230 (3):699-705;Harker, C. T., and P. M. Vanhoutte. 1988. Cooling the central ear arteryof the rabbit: myogenic and adrenergic responses. J Pharmacol Exp Ther245 (1):89-93; anal Faber, J. E. 1988. Effect of local tissue cooling onmicrovascular smooth muscle and postjunctional alpha 2-adrenoceptors. AmJ Physiol. 255 (1 Pt 2):H121-30.

These results confirm that α_(2C)-ARs do not normally contribute tovasoconstriction. However, during cold-induced vasoconstriction,α_(2C)-ARs are no longer silent and mediate the remarkable cold-inducedaugmentation of α₂-AR responsiveness. These results also indicate thatα_(2C)-AR antagonists can be used to relieve the vasospastic episodesthat occur when individuals with Raynaud's Disease are exposed to coldor stress. Because α_(2C)-AR appear to be silent in the normalregulation of vascular function, selective blockade of these receptorswith an α_(2C)-AR antagonist is expected to provide a highly selectivetherapeutic intervention for this condition.

Example 2

Effect of α_(2C)-AR Antagonists on the Vascular Smooth Muscle Cells ofPatients with Scleroderma

Materials and Methods

Subject Characteristics

Skin biopsies (6 mm punch) were obtained from the same location on themedial aspect on the upper arm of patients and control subjects. Elevenpatients with diffuse cutaneous SSc were studied: nine female and twomale. Their average age was 49 years old (range 33 to 69) and they hadSSc for an average of 4 years (range 1 to 9 years from first physiciandiagnosis of SSc). SSc patients were recruited from the Johns Hopkinsand University of Maryland Scleroderma Center. All patients met theAmerican College of Rheumatology criteria for a diagnosis of SSc. Biopsyof the skin was performed in the upper arm in an area considered to havenormal skin thickness determined by clinical palpation(clinically-uninvolved skin). Patients with overlap syndromes (e.g.lupus) were excluded. Patient medications varied between individuals:anti-inflammatory /immunosuppressant, 4 patients on prednisone, 1 onmethotrexate, 1 on cyclophosphamide, and 1 on D-penicillamine;gastrointestinal, 5 on omeprazole, 3 on cisapride, 2 on ranitidine, 1 onlansoprazole; angiotensin converting enzyme inhibitors, 3 on enalapril,1 on lisinopril; angiotensin receptor antagonists, 1 on losartin, 1 onvalsartin; calcium antagonists, 2 on nifedipine, 1 on verapamil, 1 onamlodipine, 1 on diltiazem; α₁-adrenergic receptor antagonists, 1 onterazosin. Vasoconstrictor responses to the α₁-adrenergic receptor(α₁-AR) agonist, phenylephrine, were not included for the patientreceiving terazosin. Eight normal subjects were analyzed: six female andtwo males with an average age of 50 years old (range 38 to 62). Allpatients and volunteers gave informed consent and the study was approvedby the Johns Hopkins University human subjects IRB committee.

Blood Vessel Preparation

Arterioles were dissected from the deep dermal plexus of the biopsiesand any side branches tied. Subsequent histological examination revealedno structural abnormalities in SSc arterioles. The arterioles werecannulated with glass micropipettes and placed in a microvessel chamberas previously describe. The arterioles were maintained in no-flow stateat a constant transmural pressure (P_(TM))of 40 mmHg. The chamber wassuperfused with buffer solution (37° C., pH 7.4, gassed with 16% O₂-5%CO₂-balance N₂) and placed on stage of an inverted microscope forcontinuous monitoring of internal diameter using a video camera andvideo dimension analyzer.

Experimental Protocol

Vasoconstriction was assessed in response to: i) KCl (15 to 60 mM), ii)the selective α₁-AR agonist, phenylephrine (0.01 to 1 μM) (SIGMA, StLouis, Mo.), or iii) the selective α₂-AR agonist, UK 14,304 (1 to 100nM) (brimonidine,5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine, RBI, Natick,Mass.). Responses to UK 14,304 were also evaluated following denudationof the endothelium, achieved by carefully placing a wire (70μdiameter)through the vessel lumen. Endothelium-removal was confirmed by histologyand by loss of response to the endothelial stimuli acetylcholine orbradykinin.

Data Analysis

Responses were expressed as a percentage change in baseline diameter.Data is expressed as means±SEM for n number of experiments, where nequals the number of subjects from which blood vessels were studied.Concentration-effect curves were analyzed by comparing: i) maximalresponses (vasoconstriction or dilatation), and ii) the area under thecurve (AUC). Statistical evaluation of the data was performed usingpaired or unpaired t-tests. Responses were considered to bestatistically different when P was less than 0.05.

Results

Baseline Characteristics

At a P_(TM) of 40 mmHg, there was no significant difference in thediameter of control and SSc arterioles (164±15μ and 166±18μ,respectively). The arterioles did not display spontaneous constrictoractivity, and administration of vasodilator agonists (e.g. papaverine,10 μM; sodium nitroprusside, 10 μM) did not cause relaxation inunstimulated arterioles.

Constrictor Agonists

The α₂-AR agonist, UK 14,304 (1-100 nM) caused concentration-dependentconstriction that was increased in SSc compared to control arterioles(FIG. 8). The increased reactivity was associated with an increasedmaximal response to the agonist (25±5% and 67±4% constriction in controland SSc arterioles, respectively; P=0.000014). In contrast, constrictorresponses to KCl (15 to 60 mM), a receptor-independent, smooth musclestimulus were similar in control and SSc arterioles. Likewise,constriction evoked by the α₁-AR agonist, phenylephrine (0.01 to 1 μM)was not significantly different between control and SSc arterioles, with1 μM of the agonist causing constriction of 45±8% and 45±7% in controland SSc arterioles, respectively (FIG. 8).

Because increased constriction can result from diminished activity ofendothelial dilator mechanisms, the constrictor response to α₂-ARactivation was also evaluated following mechanical denudation of theendothelium. The vasoconstrictor activity of UK 14,304 was not affectedby endothelial denudation (FIG. 8) (maximal responses of 25±7% and67±10% constriction in endothelium-denuded, control and SSc arterioles,respectively).

These results demonstrate that arterioles isolated fromclinically-uninvolved skin of diffuse SSc subjects have a selectiveincrease in the reactivity of smooth muscle α₂-ARs. SSc arterioles didnot display spontaneous vasospastic activity in the absence ofstimulation, and had normal vasoconstrictor activity in response to KClor to activation of smooth muscle α₁-ARs. Therefore, SSc arterioles donot have a generalized defect in vasomotor regulation. In addition tosmooth muscle constrictor α₂-ARs, α₂-ARs can also be present onendothelial cells, with activation leading to increased production of NOand dilatation in some blood vessels. Increased constriction to α₂-ARstimulation in SSc arterioles could therefore reflect endothelialdysfunction or injury. However, endothelial dilator function, assessedwith acetylcholine and bradykinin, was similar in control and SScarterioles. Furthermore, α₂-AR constrictor activity was not altered byendothelial denudation, indicating that the increased α₂-AR reactivityresults from selective enhancement of vascular smooth muscle α₂-ARsignaling

To determine whether this increased responsiveness resulted from alteredor enhanced expression of a particular subtype of α₂ adrenergicreceptor, RT-PCR was performed on RNA obtained from the dermal arteriesof patients with scleroderma and healthy control subjects. The RT-PCRemployed primers which are specific to the genes encoding α_(2A) andα_(2C) receptors and used standard techniques. As shown in FIG. 9, thedermal arterioles of healthy, control subjects express mRNA encoding theα_(2A) receptor but lack mRNA encoding the α_(2C) adrenergic receptor.In contrast, dermal arterioles from the uninvolved skin of sclerodermapatients lack mRNA which encodes the α_(2A) adrenergic receptors, butcontain significant amounts of mRNA which encode the α_(2C) adrenergicreceptors. It is believed that this switch in expression from the α_(2A)adrenergic receptors to the α_(2C)-adrenergic receptor is the cause ofthe increased reactivity of the α₂ adrenergic receptors and theunderlying vasculopathy in scleroderma.

To determine the effect of selective α₂ AR agonists and antagonists onvasoconstriction, small dermal arterieslarterioles from healthy controlsand from SSc subjects were obtained and analyzed using themicroperfusion system described above. In order to determine theinfluence of α_(2C)-AR blockade, paired arteries from each subject wereused. In one artery of each pair, responses to UK 14,304 were determinedrepeatedly to demonstrate that the response to UK 14,304 remainedconstant with repeated exposure (time control). Indeed, in both controland SSc arteries, the response to UK 14,304 was reproducible. In theother artery of each pair, increasing concentrations of the α_(2C)-ARantagonist (MK 912) was administered before the response to UK 14,304was determined.

The results demonstrated that extremely low concentrations of theα_(2C)-AR inhibitor MK 912 (e.g. 10⁻¹¹M) reduced the vasoconstrictorresponse to α2-AR stimulation in SSc but not in control arteries. (FIG.10). This confirms that blockade of α_(2C)-AR is capable of reducing theabnormal vasoconstrictor activity of SSc blood vessels. Indeed, thecalculated dissociation constant for MK 912 (−log Kb of 11.91) confirmedthat it was acting to inhibit α_(2C)-ARs. Much higher concentrations ofMK 912 (i.e. 10⁻⁹M or 100-fold higher) were needed to inhibit responsesto UK 14,304 in control arteries. At these concentrations, MK 912 is nolonger selective for α_(2C)-ARs, and the calculated dissociationconstant (−log Kb of 9.33) confirmed that the antagonist was acting toinhibit α_(2A)-ARs. Therefore, the functional data is in agreement withthe RT-PCR data and indicates that there is a switch in receptorexpression from α_(2A)-ARs on control arteries to α_(2C)-ARs inscleroderma. It also indicates that α_(2C)-AR antagonists inhibit theabnormally high vasoconstrictor activity in scleroderma, and thereby,reverse the disease process in vitro.

What is claimed is:
 1. A method of treating a patient with a conditionthat involves vasoconstriction of the small arteries or arterioles of apart or organ of the patient's body, comprising: administering to thepatient a therapeutically effective amount of an α_(2C) receptorantagonist that selectively binds to an α_(2C) adrenergic receptor. 2.The method of claim 1 wherein the antagonist is a reversible α_(2C)adrenergic receptor antagonist.
 3. The method of claim 1 wherein thewherein the antagonist is administered prior to exposure of the subjectto cold or stress.
 4. The method of claim 1 wherein the antagonist isadministered after exposure of the subject to cold or stress.
 5. Themethod of claim 1 wherein the antagonist is administered in an oralcomposition or a topical composition.
 6. The method of claim 1 whereinthe antagonist is administered in an amount sufficient to increase bloodflow through the small arteries or arterioles of the affected organ. 7.The method of claim 1 wherein the patient is exhibiting symptoms ofRaynaud's phenomenon.
 8. The method of claim 1 wherein the patient isexhibiting symptoms of ischemia of the small arteries or arterioles ofan organ selected from the group consisting of kidney, heart, lungs,gastrointestinal tract and combinations thereof.
 9. The method of claim8 wherein the patient has scleroderma.
 10. The method of claim 8 whereinthe antagonist is administered to the patient in a pharmaceuticalcomposition that is orally ingested or inhaled by the patient orinjected into the patient.
 11. The method of claim 8 wherein the amountof antagonist administered is from 0.01 μg to about 100 mg of antagonistper kg of body weight.
 12. The method of claim 8 wherein the antagonistis administered in multiple doses.
 13. A method of reducing cold-inducedvasoconstriction of a small cutaneous artery or arteriole in at leastone organ or part of a patient's body, comprising contacting thevascular smooth muscle cells of said artery or said arteriole with anα_(2C) receptor antagonist that selectively binds to an α_(2C)adrenergic receptor.
 14. A method of reducing constriction of the smallarteries or arterioles in an organ selected from the group consisting ofheart, lung, kidney, gastrointestinal tract and combinations thereofcomprising contacting the vascular smooth muscle cells of said artery orarteriole with an α_(2C) receptor antagonist that selectively binds toan α_(2C) adrenergic receptor.
 15. The method of claim 1, wherein theantagonist is a competitive inhibitor of an α2 adrenergic receptoragonist.
 16. The method of claim 13, wherein the antagonist is acompetitive inhibitor of an α2 adrenergic receptor agonist.
 17. Themethod of claim 14, wherein the antagonist is a competitive inhibitor ofan α2 adrenergic receptor agonist.
 18. The method of claim 13, whereinthe antagonist is a competitive inhibitor of an α2 adrenergic receptoragonist.